Project Summary/Abstract Defects in the equal partitioning of chromosomes at cell division causes aneuploidy, a genetic catastrophe that results in spontaneous abortion or birth defects if it arises in the gametes and that is a major contributor to gene dosage imbalances in almost all human cancers. The centromere is the locus on each chromosome that directs accurate chromosome segregation at cell division in healthy cells. The focus of this project is to address three related major questions regarding centromeres: How are centromeres established? How are centromeres maintained over the long timescale that human biology requires? What is the relationship between the epigenetic components that define centromere location and the DNA sequences that rapidly evolve at mammalian centromeres to drive chromosome evolution? Centromeres are the chromosomal loci that confer genetic stability at cell division, but the DNA sequences typically found at the loci are paradoxically neither necessary nor sufficient for centromere function. Rather, centromeres are specified in metazoans and many other eukaryotes through an epigenetic process. Key to epigenetic centromere specification is a histone H3 variant, CENP-A, and in this proposal we now build on the major findings we made in the previous funding cycle regarding a nucleosome structural transition conferred by its close binding partner, CENP-C, the regulation of its cell cycle-coupled chromatin assembly, its role in the earliest steps in centromere formation, and defining the major oligomeric form and structural ?signature? of CENP-A nucleosomes at human centromeres. We have identified three critical areas of investigation that are now ripe for eliciting major insight at centromeres: 1) the molecular basis for how centromeres are epigenetically maintained, 2) the processes that are coupled to the genesis of a new centromere, and 3) the molecular underpinnings of centromere changes implicated in chromosome evolution. We will pursue these topics using biochemical, genomic, structural, molecular genetic, and cell biological approaches, and together these studies have the promise to generate valuable insight into the epigenetic and genetic features of the centromere that ultimately ensure stable inheritance of the genome at cell division.